**3. Genetic homogeneity**

A completely randomized experimental design was used for all experiments. Results were statistically analyzed by one-way analysis of variance (ANOVA) and Tukey's comparison of

When evaluating the different TISs in sugarcane during in vitro propagation, significant statistical differences were observed for the number and length of shoots per explant. The bioreactors with the highest number of shoots per explant were TIB, GIB and SETIS, with 38, 40 and 41 shoots/explant, respectively, followed by RITA, with 32 shoots/explant. Regarding shoot length, the bioreactors with the longest length were TIB, GIB and SETIS with 8.6, 10.7

Semiautomation of sugarcane micropropagation using TISs is a strategy to reduce production costs. The TIB, GIB and SETIS bioreactors showed good performance in the formation of the length and number of shoots; probably their size, among other factors, favors the development of explants. On the other hand, RITA, due to its limited capacity, did not allow an increase in length and number of new shoots. Commercial sugarcane micropropagation by

Values represent mean ± SE (standard error). Means with different letters per column represent statistical difference

**Table 1.** Sugarcane (*Saccharum* spp. hybrid cv. Mex 69-290 micropropagation by different temporary immersion systems

**Figure 1.** Sugarcane micropropagation by temporary immersion systems. (a) Recipient for automated temporary immersion (RITA™), (b) gravity immersion bioreactor (GIB), (c) Temporary Immersion Bioreactor (TIB) and (d) SETIS™

means test (*p* ≤ 0.05) using SPSS statistical software (version 22 for Windows).

and 9.8 cm in length, followed by RITA with 6.0 cm in length (**Table 1**).

**TIS No. of shoots/explants Shoot length (cm)** RITA 32.8 ± 0.58 b 6.0 ± 0.27 b TIB 38.8 ± 0.67 a 8.6 ± 0.43 a GIB 40.0 ± 0.55 a 10.7 ± 0.33 a SETIS 41.0 ± 0.40 a 9.8 ± 0.47 a

TISs is shown in **Figure 1**.

104 Sugarcane - Technology and Research

(Tukey, *p* ≤ 0.05).

(TISs)).

bioreactor, after 30 d of incubation.

The genetic or epigenetic variation obtained by different in vitro propagation systems is called somaclonal variation [19]; it is a problem that affects commercial micropropagation, where it is necessary to maintain the maximum genetic homogeneity of the regenerated individuals with respect to the mother plant.

The causes of somaclonal variation are not well understood and have not been fully elucidated [20]. However, some factors that determine the frequency of somaclonal variation include the in vitro regeneration system, the type and concentration of growth regulators applied, and the number of subcultures [21]. Consequently, it is important to determine the optimal number of subcultures that can be made from an explant for each sugar cultivar to be micropropagated.

Martínez-Estrada et al. [22] determined by inter-simple sequence repeat (ISSR) markers that no more than eight subcultures should be done due to the existence of polymorphism between the subcultures produced by a Temporary Immersion Bioreactor (**Figure 2**), since above eight subcultures the length and number of shoots decrease.

Genetic homogeneity and plant health are two important quality aspects that must be addressed before the seedlings are distributed outside the laboratory. According to Lal et al. [3], contamination of cultures is a severe problem that not only reduces the frequency of shoot culture initiation from the source explants but also the total number of shoots produced at various cycles of cultures. Plant tissues could also be cultured in the presence of bacterial and/ or fungal contaminants. Therefore, a phytosanitary diagnosis should be required.

**Figure 2.** Effect of subculturing on polymorphism percentage of shoots of sugarcane (cv. Mex 69–290) using Temporary Immersion Bioreactors assessed by inter-simple sequence repeat (ISSR) markers. Each bar represents the polymorphic percentage of subcultures 1–10.
